The term “transformation” is generally understood in the biotech and chemical arts to refer to a stable incorporation of a foreign DNA or RNA into a cell which results in a permanent, heritable alteration in the cell. It is well known that when new genetic material is to be introduced into a population of cells by transformation, only a certain number of the cells are successfully transformed. It is then necessary to identify the genetically transformed cells so that these cells can be separated from the non-transformed cells of the population. Identification and separation of the transformed cells has traditionally been accomplished using “negative selection”, whereby the transformed cells are able to survive and grow, while the non-transformed cells are subjected to growth inhibition or perhaps even killed by a substance which the transformed cells, by virtue of their transformation, are able to tolerate.
For example, when a population of plant cells is transformed, selection of the transformed cells typically takes place using a selection gene which codes for antibiotic or herbicide resistance. The selection gene—which in itself generally has no useful function in the transformed plant (and may in fact be undesirable in the plant) is coupled to or co-introduced with the desired gene to be incorporated into the plant, so that both genes are incorporated into the population of cells, or rather into certain of the cells in the population, since it is difficult, if not impossible, in practice to transform all of the cells. The cells are then cultivated on or in a medium containing the antibiotic or herbicide to which the genetically transformed cells are resistant by virtue of the selection gene, thereby allowing the transformed cells to be identified, since the non-transformed cells—which do not contain the antibiotic or herbicide resistance gene in question—are subjected to growth inhibition or are killed.
These negative selection methods have, however, certain disadvantages. First of all, the non-transformed cells may die because of the presence of antibiotics or herbicides in the growth medium. As a result, when the population of cells is a coherent tissue there is a risk that not only the non-transformed cells but also the transformed cells may die, due to the fact that the death of the non-transformed cells may cut off the supply of nutrients to the transformed cells or because the damaged or dying non-transformed cells may excrete toxic compounds.
Another disadvantage of negative selection is that the presence of an unnecessary gene, for example antibiotic resistance, may be undesirable. There is concern among environmental groups and governmental authorities about whether it is safe to incorporate genes coding for antibiotic resistance into plants and microorganisms. This concern is of particular significance for food plants and for microorganisms which are not designed to be used in a closed environment (e.g. microorganisms for use in agriculture), as well as for microorganisms which are designed for use in a closed environment, but which may accidentally be released therefrom.
Positive selection is a selection system whose operating principle is converse to negative selection. Rather than conferring resistance to a negative or toxic substance, positive selection involves conferring onto the transformed cell a metabolic, or other, competitive advantage over nontransformed cells. Positive selection systems identify and select genetically transformed cells without damaging or killing the non-transformed cells in the population and without co-introduction of antibiotic or herbicide resistance genes. As alluded to above, there is increasing concern that genes conferring resistance to antibiotics and/or herbicides may disperse and be incorporated into agriculturally destructive weeds and other plants, as well as pathogenic bacteria. Indeed, transgenic plants have been banned in the European Union. As a result, more and more investigative efforts are being made to develop positive selection systems for use in plants and other cell types.